testosterone production rates in rats

7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

1/8

Journal of

Clinical

Investigation

Vol.

42, No. 11,

1963

TESTOSTERONE

PRODUCTION

RATES IN

NORMAL

ADULTS

BY STANLEaY

G. KORENMAN

HILDEGARD

WILSON,

AND

MORTIMER

B. LIPSETT

(From

the Endocrinology

Branch,

National Cancer

Institnte,

Bethesda, Aid.)

(Submitted

fo r publication May 13,

1963;

a c ce pt e d J ul y 22,

1963

The

investigation

of

clinical and

physiological

problems

related to

androgen

production

has been

hampered by

lack

of an

adequate

measure

of

tes-

tosterone production.

As

one

approach

to this

problem, Finkelstein,

Forchielli, and

Dorfman

(1 ) developed

a sensitive method

for

th e meas-

urement of

free

testosterone

in

plasma.

The sub-

sequent identification

of

testosterone

in

th e

urine

(2 ) as th e glucuronoside

(3 )

provided

a unique

metabolite

for

th e estimation

of

testosterone pro-

duction

rate

by th e isotope-dilution

method. On e

such

study,

using an

isotope-derivative

method

to

quantitate

testosterone, was

briefly

reported

by Hudson,

Coghlan,

Dulmanis,

and

Ekkel (4).

We

have measured

urinary

testosterone

by

an

adaptation of

th e

fluorescence reaction

described

by Wilson

(5).

This has

facilitated th e use of the

isotope-dilution

method

for the measurement

of

testosterone

production

rates

in

man.

MATERIALS AND

METHODS

L.S.,

L.M.,

G.S.,

R.S.,

J.S.,

C.Z.,

and

M.G.

were

healthy

young adult

volunteers

C.S. was

a 27-year-old

white

woman

in

complete

remission

after

treatment

fo r

meta-

static

choriocarcinoma.

Regular

menses

had

occurred

fo r

the

6 months

before

study.

E.H. was a 27-year-o'd

Negro

woman with

normal

menstrual function

admitted

fo r treatment

of local

recurrence

of

carcinoma

of

th e

breast.

Absolute ethanol

1

was redistilled

by

th e

method

of

Peterson

and his associates

(6).

Water

was

glass-dis-

tilled

after

th e addition

of

a

few

crystals

of KMnO4.

n-Hexane,

ether,

chloroform,

and

methanol

were

prepared

as

previously

described

(7 , 8).

Ligroin

2

was

prepared

exactly

like

n-hexane;

on

redistillation,

the fraction

boiling

from

1030 C to

1060 C wa s

collected.

Benzene,

ethyl

acetate,

and

acetic

anhydride

were

redistilled.

Pyridine was

allowed

to stand overnight

over

calcium

hydride and

then

redistilled

under

anhydrous

conditions.

Sulfuric

acid,

reagent grade,

was used

as

supplied.

1

U.

S.

Industrial

Chemicals,

Inc.,

Baltimore,

Md.

2

Eastman

Kodak

P-1628,

Eastman

Kodak

Co.,

Rochester,

N.

Y.

3

Fisher

Scientific

Corp.,

Boston,

Mass.

Silica

gel

G

4

was washed twice

with

absolute

ethanol

and once

with redistilled

ethanol.

After th e

third

wash,

th e

wet

powder

was

heated

overnight in

an oven

at

1000

C

and

then stored

at room

temperature

in a

desic-

cator.

An alcohol eluate

of a 10-g sample

of the powder

should

give

no

colored

residue.

The

steroids

5

used were

obtained from

commercial

sources.

Testosterone

and testosterone

acetate

were re-

crystallized,

and th e

melting points

agreed

with re-

ported

values.

Other materials

used were human

follicle-

stimulating

hormone FSH)

(potency, 0.1

ml

1

U

NIH

FSH-S1)

contaminated

with a

small

amount

of

luteinizing

hormone, human

chorionic

gonadotropin

HCG)

,6

and testosterone-4-C

7

(7 7

, c

pe r

mg),

which

was

chromatographed

in

systems

A2

and B6

before

use.

Partition column

chromatography.

The t e ch n iq u e p re -

viously

described (7) was

modified

so that extracts

of

1.5

days'

urine could

be resolved

on a

single

column.

The

glass

tube was

48

mm i.d.

and 30 mm long

with a

55:

50

outer

joint

at

th e

top.

Solvent

systems are

shown

in

Table

I.

Seventy

g

of silica-alumina

catalyst,

used

as supplied,

was

mixed

with 43 ml

stationary

phase

A and packed

in

about 14 0 ml mobile

phase

A

previously

poured

into th e

column.

The

dried urine extract was

applied

with suc-

cessive

portions

of

1.2,

0.6,

and

0.3

ml

stationary

phase A,

each mixed

with

an

equal

volume

of mobile phase

A.

Each transfer

was

preceded

by

placing

layers

of 2,

1,

and

0.5

g

dry

silicate on th e column.

To

develop

th e

column,

th e successive

solvents

were

allowed

to

drop freely

from

a

funnel

onto

a

constant solvent

head

of

75

ml .

Th e

third

e luat e (Bl,

Table

I) was

standardized

to

contain al l

th e testosterone.

Further

eluates

were col-

lected

only

for studies

of more

polar

metabolites.

Thin-layer

chromatography.

Thin

layer plates

(2 0

X

20

cm)

were

coated

with silica

gel

G,

activated

by

heat-

in g

in

an oven

at

1000 C

fo r 40

minutes, and

stored at

room

temperature.

coating

of

50 0

A

thickness

was

used

fo r

column

effluents,

and

200

g

wa s

used

fo r

purer

f ractions.

The

developing

systems

used were

B6 ,

ben-

zene:

ethyl

acetate

(6: 4)

and

B8 ,

benzene:

ethyl

acetate

(8:

2). Appropriate

areas

were

eluted

3 times

with

2

ml

absolute

ethanol.

4

Brinkmann Instruments,

Great

Neck,

N.

Y.

5

Th e

chemical and

trivial names

of

al l

steroids

used

are

given

in

Table IV.

6

Ayerst

Laboratories,

New

York. N.

Y.

7New England

Nuclear

Corp.,

Boston,

Mass.

1753

7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

2/8

S. G. KORENMAN

H. WILSON,

AND M. B. LIPSETT

TABLE I

Solvent

systems

and

fractions

collected

from

the partition column

Volume

Volume

System Composition

Hexane

CHC13

EtOH

H20

Eluate

collected

Typical component

ml ml ml

ml

ml

A 2 CHCl3

392

8

50 50

Al

100 16-androstene-3a-ol

98 Hexane

A2 80

C1902-17-ketosteroids

B 15

CHCl3 340

60 50 50

Bi

200

Testosterone, epitestosterone,

85 Hexane

C1902-diols

B2 300

C,903-17-KS,

pregnanetriol,

5-preg-

nenetriol

C

30

CHC13

224

96 40 40

Cl

200

Tetrahydro S

70 Hexane

C2

260* Tetrahydro

E

D 60 CHCl3

160 240

50

50

Dl 250 Tetrahydro

F,

cortolone

40 Hexane

D2 150t

E

80

CHC13

40

160 25

25 E 250

Cortol

20%

Hexane

More

polar

ketols

*

After collecting 200 ml

of C2, th e column

head is allowed to run

down

for

a further 60 ml retaining

a 15-ml

head.

t

Eluates

D2 and

E contain

overlapping

components

and

are

therefore

combined.

Paper chromatography.

Whatman

1 filter paper was

washed as previously

described (8).

Chromatograms

were

equilibrated fo r at

least 3 hours

and developed fo r

16

hours in

Bush system

A2,

ligroin: methanol: water

(100: 70: 30).

Radioactive counting. Al l

counting was done

in a

Packard

Tri-Carb

liquid scintillation

spectrometer, model

314EX.

Dry

steroid

samples were

dissolved

in

5

ml

of

toluene containing

0.4%

diphenyloxazole

PPO)

and

0.005%

1,4-bis-2- (5-phenyloxazolyl)benzene

POPOP)

.8

Discriminator and

gain settings were

such

as

to

give

an

efficiency of 74% fo r

C'4. Raw urine

samples were

counted

in

th e p ol ye th er 6 11

phosphor of Davidson and

Feigelson

(9)

with

1 ml

of urine, 1 ml of water,

and

10

to

14 ml

of phosphor

assuring

a

one-phase

system

with-

out crystallization o f d io xa ne .

Quenching was

estimated

by adding

0.1 ml of

phosphor containing a known

num-

ber

of

counts

to each sample. C'4

efficiency in

this

sys-

tem

was

25%.

Sufficient

counts

were

collected to give a

SE

of

less than 5

at th e

95%o

confidence limits unless

specifically

stated.

Gas-liquid chromatography.9

A

6-foot spiral glass

column

with

a

3.4

mm

i.d.

was prepared

with 1 sili-

cone

polymer

resin

SE-30

by

th e method described

by

Haahti (10).

A Lovelock radium-foil

Argon

ionization

detector

was

operated at

1,000

v with

Argon pressure at

20 pounds

per square

inch,

resulting

in a flow rate of

25 ml pe r minute.

The column

temperature

was

2070 C,

with detector

and flash

heater

at

2500 C.

Under

these con-

ditions,

0.2

tug

of

testosterone

gave

a

peak

height

of

21

mm

with a retention

time

of 0.51

relative

to cholestane.

Fluorometric

assay.

Sulfuric

acid

reagent

was

freshly

prepared by adding

8 parts

concentrated

H.,SO4

to

2

parts

of

90

redistilled

ethanol.

Triplicate

samples

of the

8

Pilot

Chemicals,

Watertown,

Mass.

9

Carried out

in

an

apparatus

designed by th e

Glowall

Corp., Glenside,

Pa.

f ractions

to

be

assayed

were

evaporated

to

dryness

in

acid-washed

10-

X

75-mm

test tubes and

heated fo r

12

minutes

in

a

56

C water

bath after the

a dd it io n o f

0.5

ml

of

sulfuric

acid

reagent.

The

tubes

were

then

plunged

into an ice

bath and

the

samples

diluted with

0.75 ml of

95%

redistilled

ethanol

and

mixed

thoroughly

on

a

Vor-

tex

mixer.10

Fluorescence

was

determined in an

Aminco-Bowman

spectrophotofluorometer

with

a

1-P-21

RCA

photomultiplier

tube and

an

Osram xenon

lamp.

Meter

multiplier gain

was

.01.

Slit widths

were

1/32

inch

fo r

the

0.18-ml

quartz

microcuvettes.

The excita-

tion

and fluorescence

maxima were 475

mg

and

530

mgs

respectively

fo r testosterone.

Alcohol

and

reagent

blanks

always

read

less than

20%

of the

value

of

th e

lowest

standard.

A

standard

curve

was

constructed

fo r

each

assay

from

duplicate

standards

ranging

from

0.020

to

0.160

gg.

Comments

on

method. The

purification

steps

outlined

above

were

necessary

to eliminate

contaminants found

in the

silica

gel

G,

the

water,

and

th e ethanol.

A

plateau

of fluorescence intensity

was

obtained

between

70

to

90%

HSO,

concentration

and

between

8 to

16

minutes heat-

in g

time.

Although

there

was

a

rapid decay

at

room

temperature,

fluorescence

was stable fo r 2

hours

in an

ice

bath

and

was

unaffected

by

normal

illumination.

Fluorescence

wa s

linear between 0.010

and 0.750

Ag

per

sample.

Reliability of

results.

Th e

following

studies

were

per-

formed

to

demonstrate

th e

reproducibility,

accuracy,

and

sensitivity

of

th e

method.

Equal samples

of th e

same

final

testosterone

fraction

were

measured

in

1 2 s uc ce ss iv e

assays

(Table II).

According

to

the

criterion

of Grubbs

(11),

th e

disparate

value .154

may

be

discarded,

giving

a SE

of

th e

method

of

0.0066,

or

6

at

that

level.

Samples

of

final testosterone

preparations

containing

10

Scientific

Industries, Inc.,

Queens Village,

N.

Y.

1754

7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

3/8

TESTOSTERONE

PRODUCTION

RATES IN NORMAL

ADULTS

0.2

/A g

as

measured

by

fluorescence

gave

values

in

close

agreement

when

analyzed by

gas

chromatography (Ta-

ble

III).

Moreover,

in

each instance

only

th e

single

testosterone peak

wa s

seen, suggesting

purity

of

the

final fraction.

The

fluorescence

of

a number

of

steroids

including

several

with

mobilities

similar

to that of

testosterone

was

assayed

under

these conditions

(Table

IV).

The

presence

of

a double

bond

in th e

molecule

appeared

to be

a necessary

but

not

a

sufficient

requirement

for

fluores-

cence.

The

absence

of

a

characteristic

structure

for

sul-

furic

acid-induced

fluorescence has been noted

by

other

workers

(12-14).

Measurement

of testosterone production

rate.

Testos-

terone-4-C'4 (0.3

to

1

pc)

in less

than

0.5 ml

absolute

ethanol

was

taken

up

in

20

to

30 ml

of isotonic saline

in

a syringe

and

injected

intravenously.

The

syringe

was

rinsed with the patient's

blood.

Urine

was collected

fo r

3

days

and stored

at

-

14

C.

Hydrolysis

was

carried

out

with

P-glucuronidase,'1

400 U

per

ml for

72

hours

at

37 0 C,

pH

5.0.

After

acidification

to

pH

0.8

with

HSO4,

th e

urine

was continuously

extracted

with

ether

fo r 72

hours.

The neutral

extract

was

chromatographed

on

1

or

2 silicate

columns.

The

fraction

containing

tes-

tosterone

was

treated

with

digitonin

(8),

and the

super-

natant

3a-hydroxy

f raction was

chromatographed

on

three 500-,

thin-layer

plates

in

system

B6. The

testos-

terone

area

was

eluted,

acetylated

with acetic

anhydride

in pyridine,

and chromatographed

in

system

B8.

It

was

then saponified

(15)

and

chromatographed

on

paper

in

system

A2.

Testosterone

was

located

by

scanning

in

a

Nuclear-Chicago

paper strip

scanner

model

C-100 B

and,

when

possible,

by

ultraviolet

absorption.

After rechromatography

in

system B6,

samples

of

th e

testosterone

eluate

were

assayed

for fluorescence

and

counted.

When

possible,

a

portion

was

taken

fo r

gas-

liquid

chromatography.

To establish

constancy

of

SA

in

the present

studies,

each

specimen

was

reacetylated,

resaponified,

chromatographed

in

systems

B8

and

B6 ,

re-

spectively,

and

then

assayed

and

counted.

Testosterone

production

rate

was estimated

by

the use

of

the

formula:

production

rate=radioactivity

given/SA

of

urinary

tes-

tosteronue

X

days.

The coefficient

of

variation

for the

procedure

was

cal-

culated

by analysis

of

variance

after

a

logarithmic

trans-

formation

of

th e

data in Tables

VI and VII, and

a

value

of

11%

was

obtained

(16).

The

logarithmic

trans-

formation

was

needed because

sample

variance

was

pro-

portional

to th e

means

of the data

pairs.

Thus

variation

of results

beyond 22%

was

probably

not

due to

experi-

mental

error.

About

one-half of the

variance

was

due

to

C.Z.,

in whom

a

gross

discrepancy

in the second

value

was

obtained.

RESULTS

Recovery

of

administered

radioactivity.

In

5

separate

studies,

66 to

80

of the

administered

11

Ketodase,

Warner-Chilcott

Laboratories,

Morris

Plains,

N.

J.

TABLE

II

Reproducibility

of fluorometric assay

fo r testosterone

Ag Jig

Ag

Ag

.130 .114

.133

.128

.126

.128

.119

.114

.154*

.116

.127 .124

Mean

.1235

SD

.0066

*

This

value is

an

outlier at

the

99 confidence

limits

(11).

C1 4

was

excreted

within

3 days (Table

V).

Ninety-six

to

99%o

of

this radioactivity

was

ex-

creted

within

48

hours of

injection, indicating

that a

2-day urine collection is

adequate for

esti-

mation of testosterone

production

rates.

Testosterone

production

rates

(Table

VI)

ranged

from 4 to 11.8

mg per day

in

th e

men

and

were

increased

by 23, 58, and

170%

in 3

cases

after th e

administration

of 1,000 U of

HCG

fo r

5

days. There was

no

apparent

difference

be -

tween

th e base-line

values

in

th e 3

men receiving

corticosteroids

12

and th e 2

untreated

men.

T BLE III

Comparison

of

the

testosterone

content

of

purified

urine

fractions

as determined

by

gas-liquid

chromatography

and

by fluorescence

Testosterone

by

Testosterone

by gas

Patient

Period

fluorescence chromatography

ji g

ji g

L.S.

1

.20 .22

L.S.

2

.20

.20

L.M.

1

.20 .22

G.S.

1

.20

.21

G.S. 2

.20 .21

R.S.

.2 0

.19

In 4

women,

2

receiving corticosteroids,

tes-

tosterone

production

rates

ranged

from 0.94

to

2. 8

mg

daily (Table

VII). There

was

an increase

after

FSH administration

and

a doubling

of base-

line

values

when

HCG was added.

These

in-

creases

were

greater than

th e experimental

error

of

the

method

(p

7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

4/8

S. G. KORENMAN H.

WILSON,

AND

M. B. LIPSETT

TABLE

IV

Fluorogenicity

of

various steroids*

Relative

fluorescence

Chemical

name

Trivial

name

(testosterone

=

100%)

4-Androstene-1

7,-ol-3-one

Testosterone

100

4-Androstene-

1

7a-ol-3-one

Epitestosterone

100

1 -Androstene-3,17-dione

67

173-Acetoxy-4-androstene-3-one

Testosterone acetate

60

1

-Androstene-1

7,-ol-3-one

50

4-Androstene-3,

17-dione

Androstenedione 33

4-Androstene-3 ,

1705-diol

30

4-Pregnene-1ljl,1 7a,21-triol-3,20-dione

Cortisol

20

4-Pregnene-l11,21-diol-3,20-dione

Corticosterone

20

4-Pregnene-2 1

-ol-3,20-dione Desoxycorticosterone 15

5-Androstene-3 1,1

71-diol

10

4-Androstene-6,f-ol-3,17-dione

2

16-Androstene-3,1-ol

2

4-Pregnene-1

7a-ol-3,20-dione

17a-Hydroxyprogesterone

0

4-Pregnene-1

7a,21-diol-3,11,20-trione

Cortisone

0

5a-Androstane-3a-ol-

17-one

Androsterone

0

5j1-Androstane-3a-ol-

17-one

Etiocholanolone

0

5a-Androstane-3t3-ol-

17-one

Epiandrosterone 0

5-Androstene-3,3-ol-

17-one

Dehydroepiandrosterone

0

5a-Androstane-3a,1

7 3-diol

Androstanediol

0

5j3-Androstane-3a,1

7 -diol

Etiocholanediol 0

Pregnane-3a,20a-diol Pregnanediol

0

Pregnane-3a,1

7a,20a-triol Pregnanetriol

0

4-Androstene-1

1,-ol-3,17-dione

1

1,-Hydroxyandrostenedione

0

1,4-Androstadiene-

1

7,-ol-3-one

0

5a-Androstane-

1

7fl-ol-3-one

0

5,3-Androstane-1

7,6-ol-3-one

0

*

0. 2

and

0. 5

jug

of steroid were

assayed by

the method

described

in the text.

The

fluorescence

of

testosterone

is

se t

at=

100%.

DISCUSSION

Although

fluorescence

of

testosterone

in

H2SO4

has

been

noted

previously

(12-14),

our

procedure

is th e

first

quantitative

method

applicable

to

sub-

microgram

amounts

of

the

steroid.

The

reaction

is

relatively specific, although

two

potentially

contaminating steroids,

androstenedione

and

epi-

testosterone,

are also

highly fluorogenic (Table

IV).

The

finding

of

a

single

peak

on

gas-liquid

chromatography

ruled

out

the

presence

of andro-

stenedione,

which was also

separated

on

all

the

chromatographic

systems

used.

Epitestosterone,

however, and

it s

acetate migrate in B6 and B8,

respectively, just as testosterone and

its acetate.

Furthermore,

both free

steroids have the

same

retention

time in

gas-liquid

chromatography with

th e

SE-30

column.

However,

epitestosterone

has

a

mobility

1.5

times

that of testosterone in system

A2, thus ensuring adequate separation.

This

is

of

importance, since we

have

found

epitestosterone

in

th e

urine of some

of the

male subjects

in

amounts

comparable

to those of testosterone

and

have

shown

that it

is

no t derived from

testosterone

(18). Therefore

double-isotope derivative meth-

ods

that do

not

adequately separate

testosterone

TABLE

V

Urinary

excretion

of

radioactivity

following

testosterone-4-

Cl4

administration

Daily

recovery

of

radioactivity

Radioactivity recovered

Radioactivity

Patient

Period

administered

Day

1

Day

2

Day

3

Total On day

3

dpm

dpm

dpm dpm

L.S.

2

9. 5

X

106

7.1

X

10 '

6.7 X

103 75

.9

IL.M.

2 9. 5

X 10 5 6. 2

X

10 5

4. 3

X

10 3

66

.7

G.S.

1

1.1

X

10 6

7.7

X

105

8. 2

X

10 4

3. 0

X

104

80 3. 4

G.S.

2

1.0

X

10 6

7. 0

X

10 5 4.8 X 10 4

3.1

X

104

78

4. 0

C.S.

3

2.3

X

106

1.3

X

10 6

3. 3

X 105

4.5

X

10 4

74

2. 6

1

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7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

6/8

S.

G. KORENMAN,

H.

WILSON,

AND M. B.

LIPSETT

TABLE VII

Testosterone

production

rates in

four

normal

women

Treatment

Testosterone

-- -

Testosterone

Radioactivity

production

Patient

Ag e

Period

Daily

dose

Duration*

recoveredt

SA

given

ratet

years

days pg

dpm

per

pg

dOm ing per day

E.H.

27

1

Cortisone,

15

mg

5

4.1

890

2.5

X

10 6

0.94

2.7 800 1. 0

2

Cortisone,

15

mg 11 5. 6 545 2. 4

X 10 6

1.5

Human FSH, 0. 4

ml 5

.12

445$

1.8

3

Cortisone,

15

mg

15

5. 6

346

2.5

X

10 6

2. 4

Human

FSH, 0. 4 ml

9

4.5

390

2.1

HCG, 2,000 U

4

C.S.

27 1

Cortisone,

15

mg

5

1.6

705 2.3

X

10 6

1.1

.18 684

1.1

2

Cortisone, 15

mg

11

3. 0 545 2.4

X 10 6

1.4

Human FSH, 0. 4

ml 5

.20

505:

1.6

3

Cortisone, 15 mg

15 1.7 470 2. 3

X

10 6

1.7

Human, FSH 0. 4 ml

9

.027

400

1.9

HCG, 2,000

U

4

M.G.

18

9. 7 296

2.3

X

10 6

2.6

8. 5

278

2.8

C.Z.

18

3.5

348

2. 3

X

10 6

2.211

1.9 510

1.5

1.2 342

2. 2

*

The

production-rate assay

was

performed

during

the last 3

days

of each

medication

period.

t

Duplicate

values

represent

estimates

after

repetition

of

the

acetylation

and

saponification

procedure

used

to

isolate

testosterone.

SE of

counting,

7

or less.

SE

of

counting,

20%.

This

assay

was

repeated

twice because of the

poor

initial

agreement

after

duplication.

Theoretical

considerations.

The

isotope-dilu-

tion method

depends

upon

the

dilution of

the

la-

beled testosterone

by

testosterone

from

al l

sources.

When

al l

the testosterone

is

secreted

by

the

glands,

then

a

secretion

rate

is

obtained. If,

however,

a

portion

of the

testosterone is derived from

other

steroids such

as

androstenedione

as

a result

of

peripheral

metabolism,

then

the

isotope-dilution

DEHYDROEPIANDROSTERONE

TESTIS

OTHER

GLANDS

ANDROSTENEDIONE

TESTOS~trOE

r

|TESTOSTERONE

FIG.O1.NADMODEL

SYTEMFOTROTHE

PROUCTIONOANDE

r

eT~~~~HER

,,

META~~~BOLITE.S

TESTOSTERONEI

FIG. 1.

A

MODEL SYSTEM

FO R

THE PRODUCTION

AND

METABOLISM OF

TESTOSTERONE.

The

upper

testosterone

box indicates

th e

pool

of

testosterone

that

receives

tes-

tosterone

secreted

by

th e

glands

and

produced

by

th e

liver

and

other

peripheral

tissues.

A

unique

metabolite

of

testosterone is

testosterone

glucuronoside,

which

is

rap-

idly

excreted

in

th e

urine.

technique

measures

th e

total

production of tes-

tosterone

or the

production rate.

There is

considerable

evidence that

testosterone

can

be

produced

peripherally from

other steroids.

It

ha s

been shown

that the dog liver perfused

with

dehydroepiandrosterone

synthesized testosterone

(26) and that

oral

administration of

androstene-

dione

and

dehydroepiandrosterone

to

man resulted

in

higher plasma testosterone

levels

(27).

In

th e

elegant

studies

of Vande

Wiele and his

co-workers

(21), th e

contributions of the

de -

hydroepiandrosterone

and androstenedione pools

to th e

testosterone pool

were

measured and

found to be

a

significant

fraction of

th e testos-

terone

produced.

Injected, labeled

testosterone

is

thus diluted by

testosterone

secreted by

th e

glands

and

by

that

produced

in

peripheral

tissues.

Therefore th e

isotope-dilution

technique as used

in

our

studies measures th e

production

rate of

testosterone, not

it s

glandular

secretion rate.

Only

when

there

is

no

peripheral

production of

testosterone

will

th e

production rate equal the

secretion rate.

1758

7/26/2019 TESTOSTERONE PRODUCTION RATES IN RATS

7/8

TESTOSTERONE

PRODUCTION RATES

IN NORMAL

ADULTS

From

these

considerations,

th e

production

rate

should

be a

better

measure

of

th e

total andro-

gen

available

to th e

individual

than th e

secre-

tion rate. It

would

be

necessary,

in

validating

this

conclusion,

to

show

that

al l th e

testosterone

synthesized

peripherally

is

actually

returned

to

th e

plasma before

conjugation

or

metabolism

occurs.

Assuming that

testosterone

glucuronoside

is

physiologically

inactive,

one

needs

to know

th e

degree to which th e testosterone

produced by th e

peripheral metabolism of androstenedione

is

con-

jugated

before it s

entry

into

th e

general

circula-

tion.

Since

our

measurement

of testosterone

pro-

duction rate

is based on

th e

SA

of

urinary

testos-

terone

glucuronoside,

we cannot

distinguish

be -

tween the

portions

of

peripherally

derived testos-

terone t ha t e it he r

enter

the

plasma pool

or

are

conjugated

immediately.

If

this

latter

fraction

is

an

appreciable

portion

of the

urinary

testoster-

one

glucuronoside,

then

the testosterone

produc-

tion

rate

will

overestimate th e amount of

testos-

terone

reaching

th e

plasma pool.

This

problem

is

of considerable

quantitative

significance

in

view

of

the

demonstration

(21)

that

in

one female

sub-

ject

androstenedione

was

th e

major precursor

of

testosterone.

On the

basis of

these

concepts,

we

propose

a

model

system

for the

production

and

metabolism

of testosterone

(Figure

1).

The dotted

lines

outline

a

hypothetical

testosterone

pool

that

is

not

active

androgen

because

it

is either

conju-

gated

or

metabolized

before

reaching

the

plasma.

The existence

and

quantitative

significance

of

this

pool

can

be determined

only by

a

detailed

exami-

nation

of

th e

peripheral

metabolism of

andro-

stenedione.

Such studies are

in

progress.

Th e

assumptions upon

which

isotope-dilution

methods

for

secretion

rates

are based

have been

discussed

in

detail

by

Vande

Wiele,

MacDonald,

Bolte,

and

Lieberman

(20).

When

utilizing

th e

method

for the

estimation

of

production

rates,

we

assumed

that

the

injected

radioactivity

mixes

rapidly

with

th e

single

hormonal

pool

from

which

al l

of

th e excreted

metabolite

must

come.

The

validity

of

this

assumption

in

th e

testosterone

pro-

duction-rate

assay

has

been

discussed.

We have

further

assumed

that our

rechromatographed

tracer

is

pure,

that

th e

label

is

not

lost

during

me-

tabolism,

that

th e

fraction of

hormone converted

to

th e

metabolite is

constant,

and

that

testosterone

glucuronoside

is uniquely

derived from

th e

testos-

terone

pool.

The

finding that at least

66

to

80

of

th e

administered

isotope

was excreted

in

th e

urine

within 3 days

supports th e assumption

of

complete

excretion

of radioactivity,

especially since

it

has

been

shown

that

10

to 15 may appear

in

th e

stool

(19, 28).

SUM

MARY

Testosterone

production

rate has been

measured

in normal

young

men and women by

th e isotope-

dilution technique

using a fluorometric

assay

of

urinary

testosterone.

Production

rates ranged

between

4 and 11.8 mg

daily

in

five men

and

be -

tween 0. 9

and

2.8 mg daily in

four

women.

Doses of 1,000

U of HCG

to th e men for 5

days

and

2,000

U

to

th e

women

for

5

days

significantly

increased

testosterone production

rates.

The

dif-

ference

between

secretion

and

production

rates

has

been

discussed.

ACKNOWLEDGMENT

We

ar e

indebted to Drs. Raymond

Vande Wiele and

Seymour

Lieberman fo r their

discussions

and

suggestions.

We

wish

to

thank

Mr. David

Ryan and Mr. Alf

red

Bracey fo r

their excellent

technical assistance.

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